Many inherited diseases; including cystic fibrosis, diabetes, and familial hypercholesterolemia are caused by mutations that impair the folding and intracellular trafficking of ion channels, transporters and receptors that are normally expressed at the plasma membrane. There is compelling evidence demonstrating that the mutant phenotype of many of these mutants can be suppressed by treatment with pharmacological chaperones, which are small high affinity ligands that bind to and stabilize the native 3-dimensional structure of their respective targets.
Prion diseases like Scrapie (sheep), bovine spongiform encephalopathy (BSE, cattle), or Creutzfeldt-Jakob disease (CJD, human), and other neurodegenerative diseases such as Parkinson and Alzheimer, are the result of precipitated protein aggregates. On the other hand, human diseases such as cystic fibrosis and lung emphysema are caused by the rapid disappearance of crucial proteins, like the cystic fibrosis transmembrane conductance regulator (CFTR) and α-1-antitrypsin, respectively. The discovery of the degradation process for mutated and misfolded ER proteins has shed light on the molecular mechanism underlying such seemingly different diseases.
It is of great importance for the cell to regulate the individual entities of its proteome as well as to control the structural fidelity of each of its members. Proteins destined for secretion, the plasma membrane or the cell surface are translocated from the cytoplasm into the endoplasmic reticulum (ER), the central organelle for further delivery of these proteins to their site of action. Since proteins are translocated into the ER in an unfolded state, it is the primary function of this organelle to modify and fold the translocated proteins to acquire their biologically active conformation. In the ER, proteins undergo a quality control procedure that discriminates between properly folded proteins and terminally misfolded species as well as unassembled protein subunits. The misfolded polypeptides and orphan subunits are subsequently subjected to ER-associated degradation (ERAD). The ERAD process requires retrotranslocation of the malfolded proteins across the ER membrane into the cytoplasm and subsequent degradation by the proteasome. ER degradation contributes to the molecular pathogenesis of many loss- and gain-of-toxic-function disorders.
In the ER lumen, polypeptides can be modified by a large array of ER-resident chaperones and enzymes, before they can enter the secretory pathway. The major components of this process in the ER are signal peptidase, which cleaves off the signal peptide from the newly translocated proteins; the oligosaccaryl-transferase complex (OST) which carries out N-glycosylation; and protein disulfide isomerase (PDI), which participates in disulfide bond formation. The two most studied examples of chaperones that assist proteins in their folding are the Hsp70 chaperone BiP, which recognizes hydrophobic patches on proteins, and calnexin, which binds carbohydrate moieties. Proteins are allowed to exit the ER and enter the secretory pathway only when they are properly folded and modified.
The quality control mechanism works by structural rather than functional criteria. Mutations in CFTR and α-1-antitrypsin, for example, which do not perturb the biological activity of the proteins per se, lead to ER retention and elimination of the mutant molecules, thus causing disease. In a series of glycosylation events, proteins are marked during the folding process. Recognition of the carbohydrate residues on misfolded proteins determines their delivery to the elimination machinery.
Nearly all misfolded proteins are polyubiquitylated prior to degradation. It has been suggested that polyubiquitylation is necessary for retrotranslocation. Modification of the protein may occur when the N-terminus or the first lysine residue becomes accessible to the ubiquitylation machinery. Progressive polyubiquitylation may serve as a ratcheting mechanism moving the polypeptide from the retrotranslocation channel into the cytoplasm, where the long and bulky polyubiquitin chains prevent the polypeptide from slipping back into the ER. The proteasome acts after release of the ubiquitylated substrate from the ER membrane. It is currently believed that Rpn11 de-ubiquitylates the substrate after it has been threaded into the 20S channel, thereby resulting in an irreversible commitment to proteolysis. Failure to de-ubiquitylate probably causes a sterical block to further insertion of the substrate into the proteolytic core. Following release from the substrate, the polyubiquitin chain is hydrolyzed into single ubiquitin moieties which can take part in a new round of protein degradation.
Cystic fibrosis (CF), a fatal autosomal recessive genetic disease that affects over 60,000 people worldwide, is caused by mutations in CFTR. This gene encodes the cystic fibrosis transmembrane conductance regulator protein, which functions as a Cl− channel at the apical membranes of pulmonary epithelial cells. The CFTR channel is also found in certain other epithelia, such as the sweat ducts and part of the gastrointestinal tract, but lung pathology is by far the most prominent cause of clinical disease in CFTR homozygotes and compound heterozygotes. Precisely how the loss of functional, surface-expressed CFTR channels and the consequent decrease in Cl− conductance lead to CF pathogenesis is controversial. Still, the recognition that the majority of cases of CF are the result of a defect in biogenesis or intracellular trafficking of the protein, and that the mutant protein retains at least partial function, has stimulated an intensive search for therapeutic-strategies aimed at rescuing the function of the mutant CFTR.
In view of the many serious medical conditions associated with misfolded proteins, methods of high throughput screening for agents that ameliorate these conditions are of interest. The present invention addresses these issues.